PROJECT SUMMARY The prevalence of food allergy has been dramatically increasing for the last few decades. Although studies using murine models of food allergy have greatly advanced our understanding of its pathogenesis, there are still significant knowledge gaps. Histamine-releasing factor (HRF) activate mast cells and basophils in an IgE- dependent manner. As its secretion was found in body fluids during late-phase allergic reactions, HRF has been implicated in allergic diseases. However, whether HRF is involved in allergic diseases had remained enigmatic, although its molecular identity was revealed in 1995. Our 2012 study changed this situation by identifying a subset of IgE and IgG molecules as HRF receptors: mapping of the immunoglobulin (Ig) Fab- binding sites within the HRF molecule led to the discovery of HRF sequence-based inhibitors, N19 and H3 peptides, as well as a monomeric mutant HRF-2CA, all of which blocked HRF-Ig interactions; prophylactic administration of these inhibitors, which targeted mast cells, strongly reduced the incidence of allergic diarrhea and anaphylaxis, as well as the severity of intestinal inflammation in an IgE/Fc?RI (high-affinity IgE receptor)/mast cell-dependent mouse model of food allergy. HRF is present as a monomer and disulfide-linked oligomers including a dimer. HRF dimer, but not monomer, has an ability to activate IgE-primed mast cells and basophils and to enhance allergen-triggered activation of these cells. HRF oligomers increased in the small intestine of food allergic animals. Our results collectively suggest that HRF oligomers crosslink IgE-bound Fc?RI on intestinal mast cells, leading to their activation, which is required for allergen-induced maximal intestinal type 2 inflammation. Based upon these novel data, we hypothesize that HRF oligomerization and HRF-IgE interactions are two critical events to initiate and amplify intestinal inflammation in food allergy. To test this hypothesis, we will conduct food allergy experiments with novel mutant mice lacking normal HRF dimer (Aim 1), and seek to identify the enzyme system that catalyzes oligomerization of HRF (Aim 2). We will also investigate the effects of HRF oligomerization and N-glycosylation of HRF and IgEs on HRF-IgE interactions, and potential regulation of HRF-IgE interactions at the atomic level during food allergy (Aim 3). Therefore, this study will likely establish a novel paradigm that Fc?RI-mediated mast cell activation triggered by antigen is amplified by HRF oligomers that cause a heightened inflammation in food allergy.